This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The emergence of antibiotic resistance has required that new approaches be applied in order to effectively fight a host of medically relevant bacterial infections. The currently used, imprecise antibiotics need to be replaced with novel, rigorous, and safe treatments in order to combat the evolved bacterium of today. One way to destroy bacteria is to target one of their most essential processes, metabolism. The discovery of RNA structural elements, termed riboswitches, that bind cellular metabolites and control expression of essential metabolic genes provides a unique and distinct target for development of artificial agonists to fight bacterial infections. In order to rationally design and develop effective artificial agonists/antibiotics that target bacterial riboswitches, an understanding of the structural and functional details of the riboswitch-metabolite complex is essential. The aims of this grant focus on (1) probing the molecular contribution of a conserved base pair distal to the metabolite binding site within a riboswitch that binds guanine, (2) designing a crystallization construct for structural characterization of the newly discovered pre-queuosine1 riboswitch, and (3) determining the molecular interactions between the metabolite pre-queuosine1 and its riboswitch aptamer domain. The studies described here will provide atomic level detail of the interactions between riboswitches and their ligands. Structural studies of riboswitches are essential in order to gain detailed information about how the RNA interacts with its metabolite and to ultimately design non-natural metabolite analogs that can act as antibiotics. The X-ray crystallography studies described here will also have a high impact on understanding RNA-based gene regulation.